To evaluate markers for overtraining, seven male race horses were subjected to 272 days of training consisting of daily exercise bouts of either endurance running (heart rate 140/min) or interval training (maximal heart rate), both increasing in duration and intensity. An incremental exercise test was held every 4 wk, and from day 187 it was held every 2 wk. Muscle glycogen, muscle lactate, energy-rich phosphates, adrenal response to adrenocorticotropic hormone, plasma and red blood cell volumes, and a number of blood chemical variables were measured. The horses showed symptoms of weight loss, irritability, and an inability to complete the training after the intensity of the endurance exercise was increased. Test performance was not decreased. The adrenal response to adrenocorticotropic hormone was not changed during overtraining. The decline in muscle ATP concentration during maximal exercise was less during the period of staleness, whereas plasma volume, red blood cell volume, and blood chemical variables were unchanged. It was concluded that as long as exhaustive training is alternated by light exercise, overtraining is unlikely to occur. Furthermore, no single parameter can be used to detect early overtraining.